Abstract As much as 90% of HIV-1 infections occur by transmission at mucosal surfaces. Combating the earliest events in viral entry is one strategy to combat HIV infection, since it is thought that fewer virions successfully infect the host at mucosal surfaces. Vaccines that also stimulate the systemic immune system are also needed to control HIV spread once the mucosal barrier is breached. Systemic immunization by the intramuscular route can drive mucosal immune responses, but there is also data showing that mucosal immunization may better educate responses that can act at these first sites of mucosal HIV entry. We performed vaccine route comparison studies in rhesus macaques that showed that vaginal mucosal vaccination reduced SHIV-SF162P3 viral loads better than intramuscular immunization. We more recently we performed an immunogenicity study that compared the effects of vaccine routes using new replicating single- cycle adenoviruses (SC-Ads) boosted with recombinant gp140 env. Animals immunized by only the intramuscular route had lower final SHIV neutralizing antibodies in their plasma than animals immunized by intranasal alone or intranasal and intramuscular vaccinations. Intramuscular animals had high T follicular helper (Tfh) cells in the blood, but low Tfh in lymph nodes. Conversely, animals immunized by the intranasal route had high Tfh in lymph nodes, but low Tfh in the blood. Animals immunized by the intramuscular route had lower antibody-dependent cellular cytotoxicity (ADCC) activities than animals immunized by the mucosal intranasal route. These data suggest that the route of priming and boosting with gene-based and protein vaccines may markedly affect the quality and quantity of systemic and mucosal immune responses against HIV. In this project, we will build on these interesting vaccine route observations in a team consisting of groups from Mayo Clinic, MD Anderson, and the Oregon Health & Science University. We will combine our new SC-Ad vaccines with promising SOSIP protein vaccines and deliver these by a more potent synchronous co- immunization strategy. We will test how systemic vs. mucosal vaccination drives different systemic and mucosal immune responses with key analyses of effects on mucosal cell trafficking, Tfh production, and the production of functional antibodies in systemic and mucosal compartments. We will pursue these efforts in the following Specific Aims: Specific Aim 1: Optimize synchronous vaccine strategies that utilize multiple routes of immunizations to achieve potent systemic and mucosal humoral and cellular responses. Specific Aim 2: Evaluate the protective efficacy and immune correlates of synchronous systemic and mucosal vaccination against repeated low dose mucosal challenge by clade C SHIV in rhesus macaques.